A new species of the lichenised genus Anamylopsora (Baeomycetaceae, Baeomycetales) from Tengger Desert of China

Abstract The monotypic lichenised genus Anamylopsora (Baeomycetaceae, Baeomycetales), with its single species A.pulcherrima, is distributed in the arid areas of the Northern Hemisphere, including China. In this paper, we introduce another species new to science, Anamylopsorapruinosa. The new species is characterised by a densely pruinose upper surface, abundantly thick and strong rhizines and terricolous habitat. It is also strongly supported by the phylogenetic and species delimitation analyses based on nrDNA ITS sequences, in which A.pruinosa forms well-supported clade separated from A.pulcherrima.


Introduction
The monotypic genus Anamylopsora Timdal was established in 1991 (Timdal 1991), based on the species Anamylopsora pulcherrima (Vain.) Timdal. The species was previously excluded from Psora Hoffm., as Psora pulcherrima (Vain.) Elenkin, due to having, for example, a non-amyloid tholus and hymenial gelatine and is temporarily placed in the collective genus Lecidea Ach. (Timdal 1984). Together with Lecidea, the genus Anamylopsora was included in the family Lecideaceae Chevall., although it was observed to be more similar to Trapeliaceae M. Choisy ex Hertel in the ascus structure (Timdal 1991). Lumbsch et al. (1995) established a monotypic family Anamylopsoraceae in the Agyriineae (Lecanorales) based on the ascus structure, chemistry, pycnidial structure and ascoma ontogeny, comparing with all the morphologically similar or related families, such as Agyriaceae Corda, Baeomycetaceae Dumort., Icmadophilaceae Triebel, Lecideaceae and Psoraceae Zahlbr.
Anamylopsora pulcherrima is saxicolous, common in the arid areas of the Northern Hemisphere, including Asia (China, Iran, Kirgizstan, Mongolia, Nepal, Japan), Russia and U.S.A. (Davydov 2014;Inoue 2010;Moniri and Sipman 2009;Timdal 1991;Zhurbenko 2010). During our field survey in the arid region of the Northwest China, a new species of Anamylopsora was found in Tengger Desert with the characters of terricolous habitat, dense pruina and abundant rhizines. The purpose of this study is to describe the new member of the previously monotypic genus. Phylogenetic and species delimitation analyses based on nrDNA ITS sequences are also provided.

Phenotypic analysis
All the six specimens of the new species of Anamylopsora were collected from one locality in the Ningxia Hui Autonomous Region of China, close to the Inner Mongolia Autonomous Region and are preserved in the Lichen Section of Herbarium Mycologicum Academiae Sinicae (HMAS-L). A dissecting microscope (Zeiss Stemi SV11) and compound microscope (Zeiss Axioskop 2+) were used for the study of morphology and anatomy. Standardized thin-layer chromatography (TLC, solvent system C) was used for the identification of secondary metabolites (Culberson 1972;Culberson and Kristinsson 1970;Orange et al. 2001).
DNA extraction, amplification and sequencing DNA was extracted from six fresh specimens of Anamylopsora (Table 1) following the modified CTAB method (Rogers and Bendich 1988). The internal transcribed spacer of nuclear ribosome DNA (nrDNA ITS) was chosen as the genetic marker. Primers LR1 (Vilgalys and Hester 1990) and ITS1 (White et al. 1990) were used. Reactions were carried out in 50 µl reaction volume and the components used were 3 µl total DNA, 1 µl each primer (10 µM), 25 µl 2×Taq MasterMix and 20 µl ddH 2 O. PCR amplifications were carried out in a Biometra T-Gradient thermal cycler, following conditions: initial heating step for 5 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 56 °C, and 1 min 30 s at 72 °C; a final extension step of 8 min at 72 °C was added, after which the samples were kept at 4 °C. Negative controls were prepared for each amplification series. PCR products were purified using a gel purification kit (Shanghai Huashun Bioengineering Corporation, China) following the manufacturer's instructions.

Sequence alignment and phylogenetic analysis
PCR products were sequenced using the ABI 3730 XL Sequencer by Shanghai Bio-Sune Corporation of China. Except sequences of the new species, the sequences of another species in Anamylopsora, A. pulcherrima and eight species in seven genera related as outgroups, i.e. Ainoa mooreana, Anzina carneonivea, Baeomyces placophyllus, B. rufus, Phyllobaeis imbricata, Psora crenata, Trapelia coarctata and Tephromela armeniaca, were downloaded from GenBank. The sequences were aligned using ClustalW Multiple Alignment (Thompson et al. 1994) in BioEdit 7.2.5 (Hall 1999). The programme Gblocks v0.91b (Castresana 2000;Talavera and Castresana 2007) was used to delimit and remove regions of alignment uncertainty, using options for a "less stringent" se- Table 1. Specimens of Anamylopsora from China and taxa used in the phylogenetic analysis in this study.

Taxon
Voucher specimens In the ML and Bayesian analyses, substitution models for ITS were estimated using jModelTest-2.1.9 (Darriba et al. 2012;Guindon and Gascuel 2003). Based on these results, we used the TrN + I + G model with 1000 pseudoreplicates in the ML analysis and the TrN + G model in the Bayesian analysis. Two parallel Markov chain Monte Carlo (MCMC) runs were performed in MrBayes, each using 8 million generations and sampling every 1000 steps. A 50% majority-rule consensus tree was generated from the combined sampled trees of both runs after discarding the first 25% as burn-in. Tree files were visualised with FigTree v.1.4.2 (http://tree.bio. ed.ac.uk/software/figtree/). The intraspecific and interspecific genetic distances of the Anamylopsora species were also calculated and compared.

Species delimitation analyses
Two species delimitation methods were used to circumscribe species boundaries within the genus Anamylopsora -"Automatic Barcode Gap Discovery" (ABGD) (Puillandre et al. 2012) and a Bayesian implementation of the Poisson tree process model (bPTP) (Zhang et al. 2013). For ABGD we used default parameters except for using a Pmax at 0.01 and a relative gap width of 1.5, with the model Jukes-Cantor (JC69). The bPTP model is intended for delimiting species in these single-locus molecular phylogenies, and provides an objective approach for delimiting putative species boundaries that are consistent with the phylogenetic species criteria. We used the bPTP web server (http:// species.h-its.org, Zhang et al. 2013) to delimit putative species groups using the ITS topology as the input tree and implementing default settings.

Phylogenetic analysis
The aligned matrix contained 431 unambiguous nucleotide position characters for ITS. The phylogenetic tree included 10 taxa representing five families from ca. four different orders and is illustrated in Fig. 1. Anamylopsora formed a well-supported (BS=100, PP=1.00) monophyletic clade, within which the new species obviously separated from A. pulcherrima. The genetic distances (Table 2), based on nrDNA ITS sequences within Anamylopsora, showed that the intraspecific distance range was 0.00-0.01, while the interspecific distance range was 0.04-0.05, also indicating they are two different species.
Chemistry. Alectorialic and barbatolic acids. Notes. As known, Anamylopsora pulcherrima is saxicolous, growing on calciferous and non-calciferous rocks; upper surface epruinose or more rarely pruinose with more or less white pruinose margin (Timdal 1991). While the new species, A. pruinosa, is terricolous, growing directly on the surface of sandy soil, with thick and strong rhizines penetrating into the sand. On the other hand, the upper surface of A. pruinosa is densely white pruinose, occasionally very little part naked. Phylogenetic and species delimitation analyses based on ITS sequences (Fig.1) also well supported that they are two different species.

Discussion
Except for the diagnostic characters of the new species Anamylopsora pruinosa, most characters are accordant with the genus Anamylopsora, such as the habit of thallus (squamulose), type and location of apothecia (lecideine, marginal), weakly amyloid hymenium, asci with amyloid sheet, non-amyloid tholus, ascospores and conidia, and chemistry, etc. (Lumbsch et al. 1995;Timdal 1991). In addition, the phylogenetic analysis showed Anamylopsora, including the two species, to be monophyletic. The species delimitation analyses, including ABGD and bPTP, also supported A. pruinosa and A. pulcherrima as two separate species. Therefore, both the phenotypic observations and ITS sequences well supported the new species.
As the genus Anamylopsora was known to be monotypic before this study and only the species A. pulcherrima is accepted, there are, however, three synonyms, i.e. Lecidea pulcherrima (Basionym), Lecidea hedinii and Lecidea undulata (Timdal 1991). Based on the original description of L. hedinii and L. undulata (Magnusson 1940;1944), the morphological characters of L. undulata was suspected to be most similar to the new species A. pruinosa in greyish-white lobes, densely pruinose and terricolous habitat, but L. undulata has much smaller conidia (2.5-3.5 × 0.8 µm), and 'very large, reddishbrown apothecia' (Magnusson 1940), which is much different from the new species, A. pruinosa, with larger conidia (3.75-5 × 1.25-2.5 µm) and not large (0.5-2 mm diam.) and black apothecia. Especially, we could not find any fresh materials of L. undulata and the corresponding DNA sequences were unavailable. Therefore, we could not judge whether the new species A. pruinosa is exactly the synonymized L. undulata with the knowledge we have. Fresh materials corresponding to L. undulata, are needed and then it may be possible to answer this question.
In China, Anamylopsora pulcherrima has been found and reported in some arid regions, such as Inner Mongolia and Gansu (Magnusson 1940;1944;Schneider 1979), and also in Tibet (Obermayer 2004), but all these known species grow on calciferous stone, meaning that it is saxicolous. We did not find the corresponding description about whether rhizines were present in this species and we also did not find obvious rhizines through observation of the specimen deposited in F (C0090112F). However, the terricolous new species, A. pruinosa, directly grows on the surface of sandy soil, tightly adnate to the substrate by the abundant, thick and strong rhizines, forming an important type of lichen crust in the desert area, possibly contributing to sand-fixation. Previously, we generally focused on the predominant genus Endocarpon (Verrucariaceae, Verrucariales) in the Tengger Desert (Yang and Wei 2008;Zhang et al. 2017) due to their sand-fixation ability by rhizines. Comparing to Endocarpon spp., Anamylopsora pruinosa may, however, have more and greater advantages in their type of rhizines. Therefore, it is necessary to pay more attention to some other advantageous species like Anamylopsora pruinosa and try to apply them in the sand control engineering (Wei 2005) in the near future.